The purpose of this project is to gain a better understanding of the genetic structure of HIV-1 populations in infected persons with the long-term goal of predicting the genetic behavior of HIV-1 before and after initiation of antiretroviral therapy. Drug-resistant mutants, like all HIV-1 variants, arise from a highly diverse virus population formed as the result of high mutation rate (3-5 x 10-5) and rapid virus replication (generation time ~1.1 days). Although generation of mutations in HIV-1 has been studied in vitro, the mechanisms by which new mutants spread and become fixed or extinct in the virus population in vivo are poorly understood. Determining the size of the replicating HIV-1 population is also a critical step in predicting the behavior of HIV-1 populations. We have designed a clinical protocol and a laboratory strategy to investigate the replicating size and genetic structure of HIV-1 populations in vivo. Patients enrolled in this study (Protocol 00-I-0110) have frequent plasma sampling, enabling us to investigate HIV-1 variation over a period spanning ~500 viral generations. Patients are followed up after initiation of antiretroviral therapy, which allows study of the impact of treatment on HIV-1 population size and genetic structure. The study began enrolling in May 2000 and the 20 patients enrolled thus far have had more than 500 patient visits. The Virology Core of the HIV Drug Resistance Program has developed a limiting dilution RT-PCR amplification technique to obtain p6-protease and reverse transcriptase (RT) sequences derived from 20-50 individual HIV-1 genomes in a single plasma sample. Initial findings from these data suggest that the replicating population size of HIV-1 in vivo is large. We have also collaborated with NIH investigators and area physicians to obtain specimens from patients with recent HIV-1 infection, to investigate the development of HIV-1 sequence diversity. We are now extending these studies through collaborative arrangements with AIDS Clinical Trials Group (ACTG) investigators to study HIV-1 genetic variability in patients with multiple drug resistance before and after undergoing salvage therapy. To facilitate these studies, we have also developed new techniques to estimate the frequency of rare, drug-resistant HIV-1 variants (<1/104). This highly sensitive and quantitative assay will complement population studies and will be used to study emergence of resistance mutations over time.